Cancers are among the most common causes of death in developed countries. Despite continuing advances, the existing treatments exhibit undesirable side effects and limited efficacy. Identifying new effective cancer drugs is a continuing focus of medical research.
DNA in eukaryotic cells is tightly complexed with proteins (histones) to form chromatin. Histones are small, positively charged proteins that are rich in basic amino acids, which contact the negatively charged phosphate groups of DNA. There are five main classes of histones: H1, H2A, H2B, H3, and H4. Histones are synthesized during the S phase of the cell cycle, and newly synthesized histones enter the nucleus to become associated with DNA.
The amino acid side chains of histones may be modified by post-translational addition of methyl, acetyl, or phosphate groups, neutralizing the positive charge of the side chain, or converting it to a negative charge. For example, lysine and arginine groups may be methylated, lysine groups may be acetylated, and serine groups may be phosphorylated. Methylation, acetylation, and phosphorylation of amino termini of histones that extend from the nucleosomal core affect chromatin structure and gene expression. Spencer, et al., Gene, 1999, 240, 11-12.
Acetylation and deacetylation of histones is associated with transcriptional events leading to cell proliferation and/or differentiation. Regulation of the function of transcriptional factors is also mediated through acetylation. The acetylation status of histones is correlated with the transcription of genes. Histone acetylases (e.g., histone acetyltransferases (HAT)) and deacetylases (histone deacetylases or HDACs), which regulate the acetylation state of histones have been identified in many organisms and have been implicated in the regulation of numerous genes. In general, histone acetylation is associated with transcriptional activation, whereas histone deacetylation is associated with gene repression. Histone deacetylases (HDACs) repress gene transcription by modulating histone acetylation. Some non-histone proteins, many of which are transcription factors, are also substrates of HDACs.
A growing number of histone deacetylase isoforms have been identified. The histone deacetylase family is subdivided into three categories based on sequence similarity to the yeast proteins RPD3 (class I: HDAC1, 2, 3, and 8), HDA1 (class II: HDAC4, 5, 6, 7, 9, and 10), and SIR2 (class III), while HDAC11 (class IV) shares some similarity to class I and class II, and can be considered to lie at the boundary between the two classes. The class I HDACs includes HDAC1, HDAC2, HDAC3, and HDAC8, which exhibit high sequence identity and similar domain organization, and are similar to the yeast RPD3 protein factor involved in gene transcription regulation. Class II HDACs, HDAC4, HDAC5, HDAC6, HDAC7, HDAC9, and HDAC10, are similar to yeast histone deacetylase, a complex with the active part carried by the HDA1 catalytic subunit. A third class of deacetylases (class III) (sirtuins 1-7) includes the SIR2 (silent information regulator)-like family of NAD-dependent deacetylases.
Although HDACs are involved in many cellular functions, such as cell cycling and apoptosis, the best-characterized function of Class I and II HDACs is transcriptional repression. Histone deacetylases function as part of large multiprotein complexes, which are tethered to the promoter and repress transcription. Transcriptional repression is directly associated with the recruitment of multiprotein complexes containing histone deacetylases. Well characterized transcriptional repressors such as MAD, nuclear receptors and YY1 associate with histone deacetylase complexes to exert their repressor function. Histone deacetylases have been found in association with multiprotein complexes, with some distinctions between class I and class II deacetylases. For example, class I but not class II histone deacetylases are found in association with a mouse protein mSin3a which is known to bind to MAD (a Sin3/HDAC complex), and in association with multiprotein complexes known as NuRD/Mi2/NRD. On the other hand, class II HDACs are involved in shuttling between the nucleus and the cytoplasm. However, both class I and class II HDACs possess well-conserved deacetylase core domains of approximately 400 amino acids and apparently identical zinc-dependent catalytic machinery. Class III HDACs require nicotinamide-adenine dinucleotide as a cofactor, and, at least in yeast, sense the metabolic state and age of the cell. A mammalian homolog of SIR2, SirT1, deacetylates p53, altering its function as an apoptotic protein, and another, SirT2, is a microtubule deacetylase.
Aberrant histone deacetylase activity and/or levels are believed to be associated with a variety of different disease states including, but not limited to cell proliferative diseases and conditions such as leukemia, melanomas/squamous cell carcinomas, breast cancer, prostrate cancer, bladder cancer; lung cancer, ovarian cancer and colon cancer. Histone deacetylase inhibitors exhibit various beneficial anticancer effects on cancer cells, including inducing cellular differentiation, up-regulating tumor suppressor gene expression, reducing tumor growth, inducing apoptotic cell death, and inhibiting angiogenesis. In addition to their direct effects, histone deacetylase inhibitors also enhance the beneficial effects of other agents by sensitizing cancer cells to the effect of other chemotherapeutic agents or the effects of radiation.
As a result of these beneficial effects, the development of novel histone deacetylase inhibitors as potential novel anticancer agents has been a topic of considerable research interest. For example, hydroxamic acid-containing inhibitors have been made which are high affinity reversible inhibitors of both class I and II HDACs. Trichostatin A (TSA) ((R,2E,4E)-7-(4-(dimethylamino)phenyl)-N-hydroxy-4,6-dimethyl-7-oxohepta-2,4-dienamide) was one of the first histone deacetylase inhibitors to be described and is widely used as a reference in research. The recently approved cancer drug suberoylanilide hydroxamic acid (SAHA) is also of this class, which also includes sulfonamides such as oxamflatin ((E)-N-hydroxy-5-(3-(phenylsulfonamido)phenyl)pent-2-en-4-ynamide), a compound with demonstrated anti-tumor activity, and belinostat (PXD101) ((E)-N-hydroxy-3-(4-(N-phenylsulfamoyl)phenyl)acrylamide), which inhibited growth of human cisplatin-resistant ovarian tumor xenografts of cells. Other hydroxamic-acid-sulfonamide inhibitors of histone deacetylase are described in: Lavoie, et al., Bioorg. Med. Chem. Lett., 2001, 11, 2847-50; Bouchain, et al., J. Med. Chem., 2003, 846, 820-830; Bouchain, et al., Curr. Med. Chem., 2003, 10, 2359-2372; Marson, et al., Bioorg. Med. Chem. Lett., 2004, 14, 2477-2481; Finn, et al., Helv. Chim. Acta, 2005, 88, 1630-1657; WO2002030879; WO2003082288; WO20050011661; WO2005108367; WO2006123121; WO2006017214; WO2006017215; US2005/0234033. Other structural classes of histone deacetylase inhibitors include short chain fatty acids, cyclic peptides, and benzamides. Acharya, et al., Mol. Pharmacol., 2005, 68, 917-932.